US5466597A - Bacillus thuringiensis strains and their genes encoding insecticidal toxins - Google Patents

Bacillus thuringiensis strains and their genes encoding insecticidal toxins Download PDF

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US5466597A
US5466597A US07/952,755 US95275592A US5466597A US 5466597 A US5466597 A US 5466597A US 95275592 A US95275592 A US 95275592A US 5466597 A US5466597 A US 5466597A
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bti109p
bti260
gene
protoxin
strain
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Marnix Peferoen
Bart Lambert
Katrien Van Audenhove
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Bayer CropScience NV
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Plant Genetic Systems NV
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/075—Bacillus thuringiensis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/832—Bacillus

Definitions

  • This invention relates to two new strains of B. thuringiensis (the "BtI109P strain” and the “BtI260 strain”), each of which produces crystallized proteins (the “BtI109P crystal proteins” and the “BtI260 crystal proteins”, respectively) which are packaged in crystals (the "BtI109P crystals” and the “BtI260 crystals", respectively) during sporulation.
  • the BtI109P and BtI260 strains were deposited under the provisions of the Budapest Treaty at the Deutsche Sammlung Fur Mikroorganismen and Zellkulturen (“DSM”), Mascheroder Weg lB, D-3300 Braunschwe ig, Federal Republic of Germany, under accession numbers 5870 and 5871, respectively, on Apr. 4, 1990.
  • This invention also relates to an insecticide composition that is active against Coleoptera and that comprises the BtI109P or BtI260 strain, as such, or preferably the BtI109P or BtI260 crystals, crystal proteins or the active component(s) thereof as an active ingredient.
  • This invention further relates to:
  • BtI109P gene from the genome of the BtI109P strain, which encodes an insecticidal protein (the "BtI109P protoxin") that is found in the BtI109P crystals;
  • BtI260 gene from the genome of the BtI260 strain, which encodes an insecticidal protein (the "BtI260 protoxin”) that is found in the BtI260 crystals.
  • the BtI109P and BtI260 protoxins are the proteins that are produced by their respective BtI109P and BtI260 strains before being packaged into their respective BtI109P and BtI260 crystals.
  • This invention still further relates to the "BtI109P toxin" and the "BtI260 toxin” which can be obtained (e.g., by trypsin digestion) from the BtI109P protoxin and the BtI260 protoxin, respectively.
  • the BtI109P and BtI260 toxins are insecticidally active proteins which can be liberated from the BtI109P crystals and the BtI260 crystals, respectively, produced by the BtI109P strain and the BtI260 strain, respectively.
  • Each toxin has a high activity against Coleoptera.
  • the BtI109P and BtI260 toxins are believed to represent the smallest portions of their respective BtI109P and BtI260 proteins which are insecticidally effective against Coleoptera.
  • This invention yet further relates to a chimaeric gene that can be used to transform a plant cell and that contains:
  • btI109P or btI260 gene part a part of the btI109P or btI260 gene (the "insecticidally effective btI109P or btI260 gene part) encoding an insectidicidally effective portion of the respective BtI109P or BtI260 protoxin, preferably a truncated part of the btI109P or btI260 gene (the "truncated btI109P or btI260 gene") encoding just the respective BtI109P or BtI260 toxin;
  • btI109p or btI260 chimaeric gene This chimaeric gene is hereinafter generally referred to as the "btI109p or btI260 chimaeric gene.”
  • the insecticidally effective btI109P or btI260 gene part is present in the btI109P or btI260 chimaeric gene as a hybrid gene comprising a fusion of the truncated btI109P or btI260 gene and a selectable marker gene, such as the neogene (the "btI109P-neo or btI260-neo hybrid gene”) encoding a BtI109P-NPTII or BtI260-NPTII fusion protein.
  • a selectable marker gene such as the neogene (the "btI109P-neo or btI260-neo hybrid gene") encoding a BtI109P-NPTII or B
  • This invention also relates to:
  • a cell (the "transformed plant cell") of a plant, such as potato or corn, the nuclear genome of which is transformed with the insecticidally effective btI109P or btI260 gene part;
  • a plant (the "transformed plant") which is regenerated from the transformed plant cell or is produced from the so-regenerated plant, the nuclear genome of which contains the insecticidally effective btI109P or btI260 gene part and which is resistant to Coleoptera.
  • This invention still further relates to a B. thuringiensis ("Bt") strain transformed, preferably by electroporation, with a vector-carrying all or part of the btI109P or btI260 gene.
  • Bt B. thuringiensis
  • B. thuringiensis is a gram-positive bacterium which produces endogenous crystals upon sporulation. The crystals are composed of proteins which are specifically toxic against insect larvae.
  • pathotype A that is active against Lepidoptera, e.g., caterpillars
  • pathotype B that is active against certain Diptera, e.g., mosquitos and black flies
  • pathotype C that is active against Coleoptera, e.g., beetles (Ellar st al, 1986).
  • Bt tenebrionis U.S. Pat. No. 4,766,203; European patent publication (“EP") 149,162), as Bt M-7 or Bt San Diego (EP 213,818; U.S. Pat. No. 4,771,131) and as BtS1 (European patent application (“EPA”) 88402115.5).
  • EP European patent publication
  • BtPGSI208 and BtPGSI245 Two other strains toxic to Coleoptera, BtPGSI208 and BtPGSI245, have also been described (PCT publication WO 90/09445).
  • the two new Bt strains of pathotype C i.e., the BtI109P and BtI260 strains.
  • a plant cell genome is transformed with the insecticidally effective btI109P or btI260 gene part, preferably the truncated btI109P or btI260 gene. It is preferred that this transformation be carried out with the btI109P or btI260 chimaeric gene.
  • the resulting transformed plant cell can be used to produce a transformed plant in which the plant cells in some or all of the plant tissues: 1) contain the insecticidally effective btI109P or btI260 gene part as a stable insert in their genome and 2) express the insecticidally effective btI109P or btI260 gene part by producing an insecticidally effective portion of its respective BtI109P or BtI260 protoxin, preferably its respective BtI109P or BtI260 toxin, thereby rendering the plant resistant to Coleoptera.
  • the transformed plant cells of this invention can also be used to produce, for recovery, such insecticidal Bt proteins.
  • a process for rendering a plant resistant to Coleoptera by transforming the plant cell genome with the insecticidally effective btI109P or btI260 gene part, preferably the truncated btI109P or btI260 gene.
  • the plant cell be transformed with the btI109P or btI260 chimaeric gene.
  • BtI109P and BtI260 protoxins the insecticidally effective portions of such protoxins and the BtI109P and BtI260 toxins, as well as the btI109P and btI260 genes, the insecticidally effective btI109P and btI260 gene parts, the truncated btI109P and btI260 genes and the chimaeric btI109P and btI260 genes.
  • a Bt strain is transformed, preferably by electropotation, with a vector carrying all or part of the btIlO9P or btI260 gene encoding all or an insecticidally effective portion of the BtI109P or BtI260 protoxin.
  • insecticidal composition against Coleoptera and a method for controlling Coleoptera with the insecticidal composition, wherein the insecticidal composition comprises the BtI260 or BtI109P strain, crystals, crystal proteins, protoxin, toxin and/or insecticidally effective protoxin portions.
  • FIG. 1 shows total protein patterns by SDS-PAGE of sporulated BtI109P, BtI260, BtS1 and BtPGSI208 Bacillus cultures.
  • FIG. 2 shows the hybridization pattern under low stringency conditions of EcoRI digested total DNA from strains BtS1, BtPGSI208BtI109P and BtI260 with a fragment of the bt13 gene from BtS 1 as a probe.
  • the BtI109P and BtI260 protoxins can be isolated in a conventional manner from, respectively, the BtI109P strain, deposited at the DSM under accession number 5870, and the BtI260 strain, deposited at the DSM under accession number 5871.
  • the BtI109P and BtI260 crystals can be isolated from sporulated cultures of their respective strains (Mahillon and Delcour, 1984), and then, the respective protoxins can be isolated from these crystals according to the method of Hofte et al (1986).
  • the protoxins can be used to prepare monoclonal or polyclonal antibodies specific for these protoxins in a conventional manner (Hofte et al, 1988 ).
  • the Bt1109P toxin can then be obtained by protease digestion (e.g., by trypsin digestion) of the BtI109P protoxin.
  • the BtI260 toxin can be obtained by protease digestion (e.g., by trypsin digestion) of the BtI260 protoxin.
  • the btI109P and btI260 genes can also be isolated from their respective strains in a conventional manner.
  • the btI109P or btI260 gene can be identified in its respective BtI109P or BtI260 strain, using the procedure described in patent application Ser. No. 821,582 and in EPA 86300291.1 and EPA 88402115.5 (which are incorporated herein by reference).
  • the btI109P and btI260 genes are each identified by: digesting total DNA from their respective BtI109P and BtI260 strains with one or more restriction enzymes; size fractionating the DNA fragments, so produced, into DNA fractions of 5 to 10 Kb; ligating such fractions to cloning vectors; transforming E. coli with the cloning vectors; and screening the clones with a suitable DNA probe.
  • the DNA probe can be constructed: 1) from a highly conserved region of a bt gene which encodes another crystal protoxin against Coleoptera such as: the bt13gene described in EPA 88402115.5 and by Hofte et al (1987); or 2 ) on the bas is of the N-terminal amino acid sequence of the protoxin encoded by the respective btI109P or btI260 gene, which sequence can be determined by gas-phase sequencing of the immobilized protoxin (EPA 88402115.5).
  • the 5 to 10 kB fragments prepared from total DNA of the BtI109P or BtI260 strain, can be ligated in suitable expression vectors and transformed in E. coli. The clones can then be screened by conventional colony immunoprobing methods (French et al, 1986) for expression of the BtI109P or BtI260 toxin with monoclonal or polyclonal antibodies raised against the toxin.
  • btI109P and btI260 genes can then each be sequenced in a conventional manner (Maxam and Gilbert, 1980) to obtain the DNA sequences. Hybridizations in Southern blots indicate that these genes are different from previously described genes encoding protoxins and toxins with activity against Coleoptera (Hofte and Whiteley, 1989).
  • An insecticidally effective part of each of the genes, encoding an insecticidally effective portion of its protoxin, and a truncated part of each of the sequenced genes, encoding just its toxin, can be made in a conventional manner from each gene after the gene has been sequenced.
  • the amino acid sequences of the BtI109P and BtI260 protoxins and toxins can further be determined from the DNA sequences of their respective btI109P and btI260 genes and truncated btI109P and btI260 genes.
  • an insecticidally effective part or "a part” of the btI109P or btI260 gene is meant a DNA sequence encoding a polypeptide which has fewer amino acids then the respective BtI109P or BtI260 protoxin but which is still toxic to Coleoptera.
  • Such a part of the btI109P or btI260 gene can encode a BtI109P or BtI260 protoxin which has been truncated towards at least one trypsin cleavage site of the protoxin patent application Ser. No. 821,582; EPA 86300291.1).
  • restriction sites can be introduced, flanking each gene or gene part. This can be done by site directed mutagenesis, using well-known procedures (Stanssens et al, 1987; Stanssens et al, 1989).
  • the insecticidally effective btI.109p or btI260 gene part encoding an insecticidally effective portion of its respective BtI109P or BtI260 protoxin, can be stably inserted in a conventional manner into the nuclear genome of a single plant cell, and the so-transformed plant cell be used in a conventional manner to produce a transformed plant that is insect-resistant.
  • a disarmed Ti-plasmid containing the insectidicidally effective btI109P or btI260 gene part, in Agrobacterium tumefaciens can be used to transform the plant cell, and thereafter, a transformed plant can be regenerated from the transformed plant cell using the procedures described, for example, in EP 116,718 and EP 270,822, PCT publication WO 84/02,913, EPA 87400544.0 and Gould et al. (1991) (which are also incorporated herein by reference).
  • Preferred Ti-plasmid vectors each contain the insecticidally effective bt/109P or btI260 gene part between the border sequences, or at least located to the left of the right border sequence, of the T-DNA of the Ti-plasmid.
  • other types of vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example, in EP 233,247), pollen mediated transformation (as described, for example, in EP 270,356, PCT publication WO 85/01856, and U.S. Pat. No. 4,684,611), plant RNA virus-mediated transformation (as described, for example, in EP 67,553 and U.S. Pat. No.
  • the resulting transformed plant can be used in a conventional plant breeding scheme to produce more transformed plants with the same characteristics or to introduce the insecticidally effective IbtI109P or btI260 gene part in other varieties of the same or related plant species.
  • Seeds, which are obtained from the transformed plants contain the insecticidally effective btI109P or btI260 gene part as a stable genomic insert.
  • Cells of the transformed plant can be cultured in a conventional manner to produce the insecticidally effective portion of the respective BtI109P or BtI260 protoxin, preferably the respective BtI109P or BtI260 toxin, which can be recovered for use in conventional insecticide compositions against Coleoptera (patent application Ser. No. 82 1,582 ; EPA 86300291.1.).
  • the insecticidally effective btI109P or btI260 gene part preferably the truncated btI109P or btI260 gene, is inserted in a plant cell genome so that the inserted part of the gene is downstream (i. e., 3 ') of, and under the control of, a promoter which can direct the expression of the gene part in the plant cell. This is preferably accomplished by inserting the btI109P or btI260 chimaeric gene in the plant cell genome.
  • Preferred promoters include: the strong constitutive 35s promoters (the "35S promoters") of the cauliflower mosaic virus of isolates CM 1841 (Gardner et al, 1981), CabbB-S (Franck et al, 1980) and CabbB-JI (Hull and Howell, 1987); and the TR1 ' promoter and the TR2 ' promoter (the “TR1 ' promoter” and “TR2 ' promoter", respectively) which drive the expression of the 1' and 2' genes, respectively, of the T-DNA (Velten et al, 1984).
  • the TR1 ' promoter and the TR2 ' promoter the TR1 ' promoter and the TR2 ' promoter
  • a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant (e.g., leaves and/or roots) whereby the inserted btI109P or btI260 gene part is expressed only in cells of the specific tissue(s) or organ(s).
  • the btI109P or btI260 gene part could be selectively expressed in the leaves of a plant (e.g., potato, corn, oilseed rape and rice) by placing the gene part under the control of a light-inducible promoter such as the promoter of the ribulose-1,5-bisphosphate carboxylase small subunit gene of the plant itself or of another plant such as pea as disclosed in patent application Ser. No. 821,582 and EPA 86300291.1.
  • a promoter whose expression is inducible (e.g., by temperature or chemical factors).
  • the insecticidally effective btI109P or btI260 gene part is inserted in the plant genome so that the inserted part of the gene is upstream (i.e., 5') of suitable 3' end transcription regulation signals (i.e., transcript formation and polyadenylation signals).
  • suitable 3' end transcription regulation signals i.e., transcript formation and polyadenylation signals.
  • Preferred polyadenylation and transcript formation signals include those of the octopine synthase gene (Gielen et al, 1984) and the T-DNA gene 7 (Velten and Schell, 1985), which act as 3'-untranslated DNA sequences in transformed plant cells.
  • the insecticidally effective btI109P or btI260 gene part be inserted in the plant genome in the same transcriptional unit as, and under the control of, the same promoter as a selectable marker gene.
  • the resulting hybrid btI109P or btI260-marker gene will, thereby, be expressed in a transformed plant as a fusion protein (patent application Ser. No. 821,582; EPA 86300291.1; Vaeck et al, 1987).
  • This result can be preferably accomplished by inserting a btI109P or btI260 chimaeric gene, containing the marker gene, in the plant cell genome. Any conventional marker gene can be utilized, the expression of which can be used to select transformed plant cells.
  • a suitable selectable marker gene is an antibiotic resistance gene such as the neo gene coding for kanamycin resistance (Reiss et al, 19847 EPA 87400544.0; patent application Ser. No. 821,582; EPA 86300291.1).
  • the insecticidally effective btI109P or btI260 gene part and the marker gene are expressed in a transformed plant as a fusion protein (patent application Ser. No. 821,582; EPA 86300291.17 Vaeck et al, 1987).
  • All or an insecticidally effective part of the btI109P and btI260 genes, encoding Coleopteran toxins, can also be used to transform gram-positive bacteria, such as a B. thuringiensis which has insecticidal activity against Lepidoptera or Coleoptera. Thereby, a transformed Bt strain can be produced which is useful for combatting both Lepidopteran and Coleopteran insect pests or combatting additional Coleopteran insect pests. Transformation of a bacteria with all or part of the btI109P or btI260 gene, incorporated in a suitable cloning vehicle, can be carried out in a conventional manner, preferably using conventional electropotation techniques as described in PCT patent application PCT/EP89/01539, filed Dec. 11, 1989.
  • the BtI109P or BtI260 strain also can be transformed with all or an insecticidally effective part of one or more foreign Bt genes such as: the bt2 gene (patent application Ser. No. 821,5827 EPA 86300291.1) or another Bt gene coding for all or an insecticidally effective portion of a Bt protoxin active against Lepidoptera; and/or the bt13 gene (EPA 88402115.5) or another Bt gene, such as the bt.PGSI208 gene or btPGSI245 gene (EPA 89400428.2; PCT publication WO 00/09445), coding for 811 or an insecticidally effective portion of a Bt protoxin active against Coleoptera.
  • a transformed Bt strain can be produced which is useful for combatting an even greater variety of insect pests, e.g., Lepidoptera and/or additional Coleoptera. Transformation of the BtI109P or BtI260 strain with 811 or part of a foreign Bt gene, incorporated in a conventional cloning vector, can be carried out in a well known manner, preferably using conventional electropotation techniques (Chassy et al, 1988).
  • Each of the BtI109P and BtI260 strains can be fermented by conventional methods (Dulmage, 1981) to provide high yields of cells. Under appropriate conditions which are well understood (Dulmage, 1981), the BtI109P and BtI260 strains each sporulate to provide their respective BtI109P and BtI260 crystal proteins in high yields.
  • the BtI109P and BtI260 strains, crystals, protoxins, toxins and/or insecticidally effective portions, preferably their protoxins, can each be used as the active ingredient in an insecticide composition used to control insect pests belonging to the order of Coleoptera.
  • the BtI109P or BtI260 crystals can be isolated from sporulated cultures of the BtI109P or BtI260 strain (Mahillon and Delcour, 1984), and then, the respective protoxin can be isolated from these crystals according to the method of Hofte et al (1986).
  • An insecticidal, particularly anti-Coleopteran, composition of this invention can be formulated in a conventional manner using the BtI109P or BtI260 strain or preferably its respective crystals, crystal proteins, protoxin, toxin and/or insecticidally effective portion of its protoxin as active ingredient(s), together with suitable carriers, diluents, emulsifiers and/or dispersants.
  • This insecticide composition can be formulated as a wettable powder, pellets, granules or dust or as a liquid formulation with aqueous or non-aqueous solvents as a foam, gel, suspension, concentrate, etc.
  • the concentration of the BtI109P or BtI260 strain, crystals, crystal proteins, protoxin, toxin and/or insecticidally effective protoxin portion in such a composition will depend upon the nature of the formulation and its intended mode of use.
  • an insecticide composition of this invention can be used to protect a potato field for 12 to 4 weeks against Coleoptera with each application of the composition. For more extended protection (e.g., for a whole growing season), additional amounts of the composition should be applied periodically.
  • a method for controlling insects, particularly Coleoptera, in accordance with this invention preferably comprises applying (e.g., spraying), to a locus (area) to be protected, an insecticidal amount of the BtI109P or BtI260 crystals, crystal proteins, protoxin, toxin or insecticidally effective protoxin portion, preferably protoxin.
  • the locus to be protected can include, for example, the habitat of the insect pests or growing vegetation or an area where vegetation is to be grown.
  • cells of the BtI109P or BtI260 strain can be grown in a conventional manner on a suitable culture medium and then lysed using conventional means such as enzymatic degradation or detergents or the like.
  • the protoxin can then be separated and purified by standard techniques such as chromatography, extraction, electrophoresis, or the like.
  • the toxin can then be obtained by trypsin digestion of the protoxin.
  • the BtI109P or BtI260 cells also can be harvested and then applied intact, either alive or dead, preferably dried, to the locus to be protected.
  • a purified BtI109P or BtI260 strain (either alive or dead) be used, particularly a cell mass that is 90.0 to 99.9% BtI109P or BtI260 strain.
  • the BtI109P or BtI260 cells#crystals, crystal proteins, protoxin, toxin, or insecticidally effective protoxin portion can be formulated in an insecticidal composition in a variety of ways, using any number of conventional additives, wet or dry, depending upon the particular use.
  • Additives can include wetting agents, detergents, stabilizers, adhering agents, spreading agents and extenders. Examples of such a composition include pastes, dusting powders, wettable powders, granules, baits and aerosol compositions.
  • Bt cells, crystals, crystal proteins, protoxins, toxins, and insecticidally effective protoxin portions and other insecticides, as well as fungicides, biocides, herbicides and fertilizers can be employed along with the BtI109P or BtI260 cells, crystals, crystal proteins, protoxin, toxin and/or insecticidally effective protoxin portion to provide additional advantages or benefits.
  • Such an insecticidal composition can be prepared in a conventional manner, and the amount of the BtI109P or BtI260 cells, crystals, crystal proteins, protoxin, toxin, and/or insecticidally effective protoxin portion employed depends upon a variety of factors, such as the insect pest targeted, the composition used, the type of area to which the composition is to be applied, and the prevailing weather conditions.
  • concentration of the BtI109P or BtI260 protoxin, insecticidally effective protoxin portion and/or toxin will be at least about 0.1% of the weight of the formulation to about 100% by weight of the formulation, more often from about 0.15% to about 0.8% weight percent of the formulation.
  • insects can be fed the BtI109P or BtI260 protoxin, toxin, insecticidally effective protoxin portion or mixtures thereof in the protected area, that is, in the area where such protoxin, toxin and/or insecticidally effective protoxin portion have been applied.
  • some insects can be fed intact and alive cells of the BtI109P or BtI260 strain or transformants thereof, so that the insects ingest some of the strain's protoxin and suffer death or damage.
  • FIG. 1- Total protein patterns by SDS-PAGE of sporulated BtI109P, BtI260, BtS1 and BtPGSI208 Bacillus cultures.
  • MW designates molecular weight markers.
  • the derived aminoacid sequence of the encoded BtI109P protoxin is presented beneath the DNA sequence.
  • the Btl109P strain was isolated from grain dust sampled in the Philippines and was deposited at the DSM on Apr. 4, 1990 under accession No. 5870.
  • the BtI260 strain was isolated from bat dung sampled in the Philippines and was deposited at the DSM on Apr. 4, 1990 under accession No. 5871.
  • Each strain can be cultivated on conventional standard media, preferably LB medium (Bacto-tryptone 10 g/l, yeast extract 5 g/l, NaCl 10 g/1 and agar 15 g/l), preferably at 28° C.
  • LB medium Bactamino-tryptone 10 g/l, yeast extract 5 g/l, NaCl 10 g/1 and agar 15 g/l
  • T 3 medium tryptone 3 g/l, tryptose 2 g/l, yeast extract 1.5 g/l, 5 mg HnCl 2 , 0.05 M Na 2 PO 4 , pH 6.8 and 1.5% agar
  • T 3 medium tryptone 3 g/l, tryptose 2 g/l, yeast extract 1.5 g/l, 5 mg HnCl 2 , 0.05 M Na 2 PO 4 , pH 6.8 and 1.5% agar
  • each of the BtI109P and BtI260 strains can also grow under facultative anaerobic conditions, but sporulation only occurs under aerobic conditions.
  • Sterilization of each strain occurs by autoclave treatment at 120° C. (1 bar pressure) for 20 minutes. Such treatment totally inactivates the spores and the crystalline BtI109P and BtI260 protoxins. UV radiation (254 run) inactivates the spores but not the protoxins.
  • BtI109P belongs to serotype H 303b, at an agglutination titre of 25,000 with Bt kurstaki.
  • BtI 260 belongs to serotype H18, at an agglutination titre of 3,200 with Bt kumamotoensis.
  • the BtI109P and BtI260 strains were grown for 48 to 72 hours at 28° C. on T 3 medium. After sporulation, the spores and crystals were harvested in phosphate buffered saline solution ("PBS" from Oxoid Ltd., Basingstroke, Hampshire, U.K.). The resulting aqueous spore-crystal suspensions were centrifuged, and the pellets were resuspended in PBS, recentrifuged and the pellet resuspended again.
  • PBS phosphate buffered saline solution
  • Example 3 Insecticidal activity of the BtI109P and BtI260 crystal proteins
  • both strains were grown for 48 to 72 hrs at 28° C. on T 3 medium. After sporulation, the spores and crystals were harvested in PBS (phosphate buffered saline). The resulting spore-crystal suspensions were centrifuged, and the pellets were resuspended, recentrifuged and the pellets again resuspended after removal of the supernatant. The pellets were incubated overnight in aqueous solutions containing 50 mM Na 2 CO 3 and 5 mM dithiotreitol. After centrifugation, the supernatants were recovered, and the protein contents of the extracts of the respective crystal proteins of the two strains were determined.
  • PBS phosphate buffered saline
  • Potato leaves were dipped either in standardized spore-crystal mixtures or in aqueous dilutions of the crystal protein solutions and then air dried for two hours. Colorado potato beetle larvae of the first instar were placed on the treated leaves, and mortality of the larvae was determined after three days. These results were compared with the mortality of larvae fed leaves treated with either spore-crystal mixtures or solubilized crystal proteins of BtSl (from DSM, accession no. 4288) which was used as a reference strain.
  • LC50 (50% lethal concentration), expressed either as ug of solubilized crystal proteins/ml solution or as the number of spore-crystals in the dip-suspension, was calculated by Probit analysis (Finney, 1971). The results, including the 95% confidence interval and the slope of the probit line, are summarized in Tables 1 and 2, below.
  • the BtI109P and BtI260 protoxins from the BtI109P and BtI260 strains respectively were detected by ELISA (Engvall and Pesce, 1978) with a polyclonal antiserum against the Bt13 coleopteran toxin (Hofte et al, 1987).
  • the btI109P and btI260 genes were identified in their respective strains by preparing total DNA of these strains and then digesting the DNA with the restriction enzymes NlaIV and EcoRI.
  • the EcoRI-digested DNA was analyzed by Southern blotting, probing with a 32 P labeled 1.46 kb PstI-EcoRV fragment from the genome of the BtS1 strain (EPA 88402115.5) containing the bt13 gene. After hybridization with the probe, the blot was washed under low stringency conditions (2XSSC, 0.1%SDS at 68° C. for 2 ⁇ 15 min) and developed. The autoradiogram (FIG. 2) shows that only the btI109P gene is related to the bt13 gene.
  • the hybridization pattern with the probe also showed that the btI109P gene was clearly different from the bt13 gene and that the genome of the BtI260 strain did not contain DNA sequences that are related to the pstI-EcoRV probe fragment of bt13 (cryIIIA) under the experimental conditions used. (FIG. 2)
  • the NlaIV-digested DNA was analyzed by Southern blotting, probing with 32 P-labeled or digoxygenin (Non-Radioactive labeling Kit, Boehringer Mannheim, Mannheim, Germany) 1.38 kb EcoRV-NcoI fragment from the genome of the BtPGSI208 strain (PCT patent application PCT/EP90/00244) containing the btPGSI208 or cryIIIB gene. After hybridization with the probe, the blot was washed under low stringency conditions (2XSSC, O.1%SDS at 68° C. for 2 ⁇ 15 min) and developed. The results (FIG. 3) show that only the btI260 gene is related to the btPGSI208 gene.
  • the hybridization pattern with the probe also showed that the btI260 gene was clearly different from the btPGSI208 gene and that the btI109P gene strain contains DNA sequences that are only distantly related to the btPGSI208 gene under the experimental conditions used (FIG. 3).
  • the gene is cut with appropriate restriction enzymes to give the truncated btI109P gene, encoding the BtI109P toxin.
  • Example 6 cloning and expression of the btI260 gene
  • total DNA from the BtI260 strain is prepared and partially digested with Sau 3A.
  • the digested DNA is size fractioned on a sucrose gradient and fragments ranging from 5 Kb to 10 Kb are ligated to the BglII--digested and BAP-treated cloning vector pECOR251 (deposited under accession no. 4711 at DSM).
  • Recombinant E.coli clones are then screened with an internal NcoI-EcoRV DNA fragment of the btPGSI208 gene (EP 382,990), as a probe, to identify clones containing the btI260 gene.
  • DNA fragments containing the btI260 gene are then sequenced (Seq. Id. no. 2) according to Maxam and Gilbert (1980).
  • the gene is cut with appropriate restriction enzymes to give the truncated btI260 gene encoding the BtI260 toxin.
  • Example 7 Construction of a btI109P-neo hybrid gene and a btI260-neo hybrid gene
  • Example 8 Insertion of the btI109P and btI260 genes the truncated btI109P and btI260 genes and the btI109P-neo and btI260-neo hybrid genes in E. coli and insertion of the truncated btI109P and btI260 genes and the btI109P-neo and btI260-neo hybrid genes in potato plants
  • cassettes each containing one of the truncated and/or hybrid genes, are each inserted in an intermediate plant expression vector (between the T-DNA border sequences of this vector), are each fused to transcript formation and polyadenylation signals in the plant expression vector, are each placed under the control of a constitutive promoter such as the promoter from cauliflower mosaic virus driving the 35S3 transcript (Hull and Howell, 1987) or the 2' promoter from the TR-DNA of the octopine Ti-plasmid (Velten et al, 1984), and are each fused to 3' end transcript formation and polyadenylation signals capable of acting in plants, such as the 3' end of the octopine synthase gene (Gielen et al, 1984).
  • a constitutive promoter such as the promoter from cauliflower mosaic virus driving the 35S3 transcript (Hull and Howell, 1987) or the 2' promoter from the TR-DNA of the octopine Ti-plasmid (Velten et al, 1984), and
  • the intermediate plant expression vectors containing the truncated btI109P and btI260 genes and the btI109P-.ne 0 and btI2 60-neo hybrid genes, are transferred into the Agrobacterium strain C 58 Cl Rif R (patent application Ser. No. 821,582; EPA 86300291.1) carrying the disarmed Ti-plasmid pGV2260 (Vaeck et al, 1987). Selection for spectinomycin resistance yields cointegrated plasmids, consisting of pGV2260 and the respective intermediate plant expression vectors.
  • Each of these recombinant Agrobacterium strains is then used to transform different potato plants (Solanum tuberosum) so that the truncated btI1.09P gene, the truncated btI260 gene, the btI109P-neo hybrid gene and the btI260-neo hybrid gene are contained in, and expressed by, different potato plant cells.
  • Example 9 Expression of the truncated btI109P and btI260 genes and the btI109P-neo and btI260-neo hybrid genes in potato plants
  • the insecticidal activity against Coleoptera of the expression products of the truncated btI109P and btI260 genes and the btI109P-neo and btI260-neo hybrid genes in leaves of transformed potato plants, generated from the transformed potato plant cells of Example 8, is evaluated by recording the growth rate and mortality of Leptinotarsa decemlineata larvae fed on these leaves. These results are compared with the growth rate of larvae fed leaves from untransformed potato plants. Toxicity assays are performed as described in EPA 88402115.5, patent application Ser. No. 821,582 and EPA 86300291.1.
  • a significantly higher mortality rate is obtained among larvae fed on leaves of transformed potato plants containing the truncated btI109P gene, the truncated btI260 gene, the btI109P-neo hybrid gene or the btI260-neo hybrid gene than among larvae fed the leaves of untransformed plants.
  • this invention is not limited to the BtI109P (DSM 5870) strain and the BtI260 (DSM 5871) strain. Rather, the invention also includes any mutant or variant of the BtI109P or BtI260 strain which produces crystals, crystal proteins, protoxin or toxin having substantially the same properties, particularly insecticidal properties, as the BtI109P or BtI260 crystals, crystal proteins, protoxin or toxin.
  • variants of the BtI109P and BtI260 strains include variants whose total protein pattern is substantially the same as the protein pattern of either the BtI109P strain or the BtI260 strain as shown in FIG. 1.
  • This invention also is not limited to potato plants transformed with the truncated btI109P or btI260 gene. It includes any monocotyledonous or dicotyledonous plant, such as tomato, tobacco, rapeseed, alfalfa, sunflowers, cotton, corn, soybeans, brassicas, sugar beets and other vegetables, transformed with an insecticidally effective btI109P or btI260 gene part.
  • Agrobacterium tumefaciens Ti-plasmids for transforming plant cells with an insecticidally effective btI109P or btI260 gene part.
  • Other known techniques for plant cell transformations such as by means of liposomes, by electropotation or by vector systems based on plant viruses or pollen, can be used for transforming monocotyledons and dicotyledons with such a gene part.
  • an insecticidally effective btI109P or btI260 gene part can be used to transform certain selected lines of corn and rice plants by methods such as are described by Fromm et al (1990), Gordon-Kamm et al (1990), Shimamoto et al (1989) and Datta et al (1990).
  • DNA sequences other than those present naturally in the BtI109P and BtI260 strains and encoding respectively the natural BtI109P and BtI260 protoxins, toxins and insecticidally effective protoxin portions can be used for transforming plants and bacteria.
  • the natural DNA sequence of these genes can be modified by: 1) replacing some codons with others that code either for the same amino acids or for other, preferably equivalent, amino acids; and/or 2) deleting or adding some codons; provided that such modifications do not substantially alter the properties, particularly the insecticidal properties, of the encoded BtI109P or BtII260 protoxin, the insecticidally effective portion of the BtI109P or BtI260 protoxin or the BtI109P or BtI260 toxin.
  • DNA recombinants containing the aforementioned DNA sequences in association with other foreign DNA particularly the DNA of vectors suitable for transforming plants and microorganisms other than E. coli, are encompassed by this invention.
  • this invention is not limited to the specific plasmids containing the btI109P and btI260 genes, or parts thereof, that were heretofore described, but rather, this invention encompasses any DNA recombinants containing a DNA sequence that is their equivalent.
  • the invention relates to all DNA recombinants that include all or part of either the btI109P gene or the btI260 gene and that are suitable for transforming microorganisms (e.g., plant-associated bacteria such as Bacillus subtilis, Pseudomonas, and Xanthomonas or yeasts such as Streptomyces cerevisiae) under conditions which enable all or part of the gene to be expressed and to be recoverable from said microorganisms or to be transferred to a plant cell.
  • transforming microorganisms e.g., plant-associated bacteria such as Bacillus subtilis, Pseudomonas, and Xanthomonas or yeasts such as Streptomyces cerevisiae

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JPH05507194A (ja) 1993-10-21
EP0528857A1 (en) 1993-03-03
US5837237A (en) 1998-11-17
ATE212667T1 (de) 2002-02-15
AU639788B2 (en) 1993-08-05
CA2079877C (en) 2001-07-31
PL295547A1 (OSRAM) 1992-10-05
DE69132913T2 (de) 2002-08-29
US5723756A (en) 1998-03-03
CA2079877A1 (en) 1991-10-27
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